Structural models of human apolipoprotein A-I: a critical analysis and review.

Human apolipoprotein (apo) A-I has been the subject of intense investigation because of its well-documented anti-atherogenic properties. About 70% of the protein found in high density lipoprotein complexes is apo A-I, a molecule that contains a series of highly homologous amphipathic alpha-helices. A number of significant experimental observations have allowed increasing sophisticated structural models for both the lipid-bound and the lipid-free forms of the apo A-I molecule to be tested critically. It seems clear, for example, that interactions between amphipathic domains in apo A-I may be crucial to understanding the dynamic nature of the molecule and the pathways by which the lipid-free molecule binds to lipid, both in a discoidal and a spherical particle. The state of the art of these structural studies is discussed and placed in context with current models and concepts of the physiological role of apo A-I and high-density lipoprotein in atherosclerosis and lipid metabolism.

Crystallization of the Mycobacterium tuberculosis cell-division protein FtsZ.

Mycobacterium tuberculosis FtsZ (MtbFtsZ), an essential protein in bacterial cell division, has been crystallized in the presence of a new inhibitor of MtbFtsZ polymerization and GTPase activity, ethyl (6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1, 4]diazepin-8-yl)carbamate (SRI-7614). Crystals of the MtbFtsZ-SRI-7614 complex (form I, 30% polyethylene glycol 4000, 0.1 M sodium citrate pH 5.6, 0.2 M NH(4)OAc, 293 K) belong to space group P6(1) or P6(5), with unit-cell parameters a = 88.78, c = 178. 02 A, and diffract to 2.3 A resolution. A second crystal form, of the GDP complex, grows in the presence or absence of Mg(2+) from PEG 4000 at 277 K or from (NH(4))(2)SO(4) at 293 K, respectively (form II, space group P6(2)22 or P6(4)22, with unit-cell parameters a = 135.02, c = 328.97 A or a = 129.30, c = 327.97 A, respectively). Complete data sets to approximately 7 A resolution have been collected from both. Exceptional form II crystals diffract to at least 4.5 A resolution. Determination of the MtbFtsZ structure may advance the design of improved inhibitors of FtsZ polymerization.

Slow polymerization of Mycobacterium tuberculosis FtsZ.

The essential cell division protein, FtsZ, from Mycobacterium tuberculosis has been expressed in Escherichia coli and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90 degrees light scattering. The mycobacterial protein reaches maximum polymerization much more slowly ( approximately 10 min) than E. coli FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg(2+) and GTP. The minimum concentration of FtsZ needed for polymerization is 3 microM. Electron microscopy shows that polymerized M. tuberculosis FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2, 3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate+ ++ (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits M. tuberculosis FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.

The two toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase isozymes form heterotetramers.

Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.

Substrate deformation in a hypoxanthine-guanine phosphoribosyltransferase ternary complex: the structural basis for catalysis.

BACKGROUND: Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are well-recognized antiparasitic drug targets. HGPRT is also a paradigmatic representative of the phosphoribosyltransferase family of enzymes, which includes other important biosynthetic and salvage enzymes and drug targets. To better understand the reaction mechanism of this enzyme, we have crystallized HGPRT from the apicomplexan protozoan Toxoplasma gondii as a ternary complex with a substrate and a substrate analog. RESULTS: The crystal structure of T. gondii HGPRT with the substrate Mg2+-PRPP and a nonreactive substrate analog, 9-deazaguanine, bound in the active site has been determined at 1.05 A resolution and refined to a free R factor of 15.4%. This structure constitutes the first atomic-resolution structure of both a phosphoribosyltransferase and the central metabolic substrate PRPP. This pre-transition state complex provides a clearer understanding of the structural basis for catalysis by HGPRT. CONCLUSIONS: Three types of substrate deformation, chief among them an unexpected C2'-endo pucker adopted by the PRPP ribose ring, raise the energy of the ground state. A cation-pi interaction between Tyr-118 and the developing oxocarbenium ion in the ribose ring helps to stabilize the transition state. Enforced substrate propinquity coupled with optimal reactive geometry for both the substrates and the active site residues with which they interact contributes to catalysis as well.

Crystal structures of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase-GMP and -IMP complexes: comparison of purine binding interactions with the XMP complex.

The crystal structures of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been determined at 1.65 and 1.90 A resolution. These complexes, which crystallize in space groups P2(1) (a = 65.45 A, b = 90.84 A, c = 80. 26 A, and beta = 92.53 degrees ) and P2(1)2(1)2(1) (a = 84.54 A, b = 102.44 A, and c = 108.83 A), each comprise a tetramer in the crystallographic asymmetric unit. All active sites in the tetramers are fully occupied by the nucleotide. Comparison of these structures with that of the xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex reported in the following article [Héroux, A., et al. (1999) Biochemistry 38, 14495-14506] shows how T. gondii HGPRT is able to recognize guanine, hypoxanthine, and xanthine as substrates, and suggests why the human enzyme cannot use xanthine efficiently. Comparison with the apoenzyme reveals the structural changes that occur upon binding of purines and ribose 5'-phosphate to HGPRT. Two structural features important to the HGPRT mechanism, a previously unrecognized active site loop (loop III', residues 180-184) and an active site peptide bond (Leu78-Lys79) that adopts both the cis and the trans configurations, are presented.

Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg(2+) ions bound: insights into the catalytic mechanism.

The crystal structure of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex has been determined at 1. 60 A resolution. This biproduct, post-transition state structure is of a T. gondii HGPRT mutant (Asp150Ala or D150A). The D150A mutant has reduced activity (k(cat) lower by 11-, 296-, and 8.6-fold for hypoxanthine, guanine, and xanthine, respectively) compared to wild-type T. gondii HGPRT. The Michaelis constants for purine bases are altered only slightly, whereas those for alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP) are lower by approximately 6.5-fold. The ternary complex crystallizes in space group C222(1) (a = 55.21 A, b = 112.25 A, and c = 144.28 A) with two subunits in the asymmetric unit; the HGPRT tetramer is completed by the application of 2-fold crystallographic symmetry. All active sites contain XMP ¿bound in a fashion similar to that of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes reported in the preceding article [Héroux, A., et al. (1999) Biochemistry 38, 14485-14494]¿, pyrophosphate, and two Mg(2+) ions. Each Mg(2+) ion is octahedrally coordinated by two terminal pyrophosphate oxygen atoms and several ordered water molecules. This structure shows how HGPRT uses two Mg(2+) ions to orient and activate the pyrophosphate moiety of PRPP for attack by a purine base, and why mutation in humans of the residue corresponding to Asp206, the only HGPRT amino acid that directly contacts the Mg(2+) ions, causes Lesch-Nyhan syndrome (HGPRT(Kinston), D193N). The Leu78-Lys79 peptide bond in the active site adopts the cis configuration, which it must to bind PRPP or pyrophosphate. The contribution of cis-trans isomerization of this peptide bond to the energetics of substrate binding and product release is discussed. A comprehensive description of the HGPRT reaction mechanism is also proposed.

Crystal structure of the ectodomain of human transferrin receptor.

The transferrin receptor (TfR) undergoes multiple rounds of clathrin-mediated endocytosis and reemergence at the cell surface, importing iron-loaded transferrin (Tf) and recycling apotransferrin after discharge of iron in the endosome. The crystal structure of the dimeric ectodomain of the human TfR, determined here to 3.2 angstroms resolution, reveals a three-domain subunit. One domain closely resembles carboxy- and aminopeptidases, and features of membrane glutamate carboxypeptidase can be deduced from the TfR structure. A model is proposed for Tf binding to the receptor.

Crystallization of truncated human apolipoprotein A-I in a novel conformation.

The crystallization of recombinant human apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, in a new crystal form is described. The fragment crystallized, residues 44-243 of native apo A-I [apo Delta(1--43)A-I], is very similar to intact native apo A-I in its ability to bind lipid, to be incorporated into high-density lipoproteins and to activate lecithin-cholesterol acyl transferase. Apo Delta(1-43)A-I crystallizes, in the presence of beta-D-octylglucopyranoside, in space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 37. 11, b = 123.62, c = 164.65 A and a diffraction limit of 3.2 A. These form II crystals grow under conditions of significantly lower ionic strength than the original form I crystals (space group P2(1)2(1)2(1), a = 97.47, b = 113.87, c = 196.19 A, diffraction limit 3.0 A). Packing arguments show that the unusual open conformation of apo Delta(1-43)A-I found in the form I crystals cannot be packed into the smaller oddly proportioned form II unit cell. Monomeric apo Delta(1-43)A-I, as either a four-helix bundle ( approximately 75 x 30 x 30 A) or an extended helical rod (approximately 150 x 20 x 20 A), can be packed into the form II unit cell. It is concluded, therefore, that apo Delta(1-43)A-I may have crystallized in one of these distinct conformations in the form II crystals.

Michellamine alkaloids inhibit protein kinase C.

Michellamines A, B, and C have shown antiviral activity against HIV-1 and HIV-2 in cell culture. They act in a complex manner by at least two reported antiviral mechanisms, inhibition of HIV reverse transcriptase and inhibition of HIV-induced cellular fusion. On the basis of their structural similarity to other protein kinase C (PKC) inhibitors, we have investigated another possible mechanism-inhibition of PKC. The michellamines were found to inhibit rat brain PKC with IC50 values in the 15-35 microM range. Michellamine B was a noncompetitive PKC inhibitor with respect to ATP with a Ki value of 4-6 microM, whereas mixed-type inhibition was observed when the peptide concentration was varied. Michellamine B inhibited the kinase domain of PKC similarly. These results indicate that the michellamines bind to the PKC kinase domain and not its regulatory domain. Molecular modeling showed that all three michellamines can bind in the active site cleft of the PKC kinase domain, to block both the ATP and the peptide substrate subsites.

Human apolipoprotein A-I: structure determination and analysis of unusual diffraction characteristics.

The crystallization and structure determination of recombinant human apolipoprotein A-I (apo A-I), the major protein component of high-density lipoprotein, is described. The fragment crystallized, residues 44-243 of native apo A-I [apo Delta(1-43)A-I], is very similar to intact native apo A-I in its ability to bind lipid, to be incorporated into high-density lipoproteins and to activate lecithin-cholesterol acyl transferase. Apo Delta(1-43)A-I crystallizes from 1.0-1.4 M sodium citrate pH 6.5-7.5 in space group P2(1)2(1)2(1), with unit-cell parameters a = 97.47, b = 113.87, c = 196.19 A (crystal form I). The crystals exhibit unusual diffraction intensity spikes and axial extinctions that are discussed in the context of the 4 A crystal structure. When flash-cooled to 100 K, the crystals diffract synchrotron radiation to 3 A resolution. Radiation sensitivity and crystal-to-crystal variation have hindered the assembly of a complete 3 A data set.

Crystal structure of truncated human apolipoprotein A-I suggests a lipid-bound conformation.

The structure of truncated human apolipoprotein A-I (apo A-I), the major protein component of high density lipoprotein, has been determined at 4-A resolution. The crystals comprise residues 44-243 (exon 4) of apo A-I, a fragment that binds to lipid similarly to intact apo A-I and that retains the lipid-bound conformation even in the absence of lipid. The molecule consists almost entirely of a pseudo-continuous, amphipathic alpha-helix that is punctuated by kinks at regularly spaced proline residues; it adopts a shape similar to a horseshoe of dimensions 125 x 80 x 40 A. Four molecules in the asymmetric unit associate via their hydrophobic faces to form an antiparallel four-helix bundle with an elliptical ring shape. Based on this structure, we propose a model for the structure of apo A-I bound to high density lipoprotein.

Studies on pig aldose reductase. Identification of an essential arginine in the primary and tertiary structure of the enzyme.

Reaction of pig muscle aldose reductase with phenylglyoxal resulted in the chemical modification of 2 arginine residues with accompanying loss of catalytic activity. The amino acid sequences of radioactive peptides resulting from the reaction of aldose reductase with [14C]phenylglyoxal followed by tryptic digestion and high performance liquid chromatography separation allowed identification of the modified arginine residues as R268 and R293. In the presence of the coenzyme NADP+, R268 is protected from modification by phenylglyoxal, while R293 becomes hyper-reactive. Phenylglyoxal modification of aldose reductase is slowed 3-fold by the presence of the coenzyme analog ADPRP; however, both arginines are still modified. These chemical modification results are in complete accord with the previously determined crystal structures of human and porcine aldose reductase complexed with NADPH, NADP+, and ADPRP. These structures indicate that R268 is located at the adenosine binding site, salt bridged to the 2'-phosphate group of NADP(H) and ADPRP. Arginine 293 is near the surface of the enzyme and is part of the C-terminal loop. In the apoenzyme or the ADPRP complex, R293 is partially protected by loop 7; upon binding NADP(H), loop 7 folds down over the coenzyme, thus exposing R293 to solvent. Our modification studies provide further evidence of the conformational change that occurs during the aldose reductase catalytic cycle.

Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110.

Structural models of human aldose reductase complexed with NADPH have revealed the apposition of C4 of the nicotinamide ring with tyrosine 48 and histidine 110, suggesting that either of these residues could function as the proton donor in the reaction mechanism. Tyrosine 48 is also part of a hydrogen-bonding network that includes lysine 77 and aspartate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing structurally conservative replacements at these sites. Enzymatic activity was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) although affinity for NADPH was unchanged. In contrast, a mutant containing asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in Km, but only about a 14-fold reduction in kcat measured with D-glyceraldehyde. Modest changes in catalytic properties were observed in the mutant containing aspartate 43 substituted with asparagine (D43N): Km for aldehyde substrates was elevated up to 17-fold, and kcat decreased less than 16-fold. However, the Kd(NADP) values for D43N were about 5 times higher than those for wild type. Mutant enzyme containing methionine substituted for lysine 77 (K77M) was up to 1,460-fold less active than the wild type. These results are consistent with Tyr-48 acting as the acid-base catalyst in human aldose reductase and confirm the importance of Asp-43, Lys-77, and His-110 to the structure and function of the active site.

The crystal structure of the aldose reductase.NADPH binary complex.

Aldose reductase is an NADPH-dependent oxidoreductase that catalyzes the reduction of a broad range of aldehydes, including glucose. Since aldose reductase has been strongly implicated in the development of the chronic complications of diabetes mellitus, much effort has been devoted to understanding the structure and mechanism of this enzyme, and many aldose reductase inhibitors have been developed as potential drugs for the treatment of these complications. We describe here the 2.75 A crystal structure of recombinant human aldose reductase (Cys-298 to Ser mutant) complexed with NADPH. This mutant displays unusual kinetic behavior characterized by high Km/high Vmax substrate kinetics and reduced sensitivity to certain aldose reductase inhibitors. The crystal structure revealed that the enzyme is a beta/alpha-barrel with the coenzyme-binding domain located at the carboxyl-terminal end of the parallel strands of the barrel. The enzyme undergoes a large conformational change upon binding NADPH which involves the reorientation of loop 7 to a position which appears to lock the coenzyme into place. NADPH is bound to aldose reductase in an unusual manner, more similar to FAD- rather than NAD(P)-dependent oxidoreductases. No disulfide bridges were observed in the crystal structure.

Crystallization and X-ray diffraction studies of a soluble form of the human transferrin receptor.

A soluble tryptic fragment of the human transferrin receptor (residues 121 to 760) has been crystallized from 2.8 M-KCl (pH 6.2) and polyethylene glycol 8000. This fragment retains the transferrin-binding activity of intact transferrin receptor. Although the trypsin treatment removes the intermolecular disulfide bonds, the receptor fragment is dimeric both under physiological conditions and at the high salt concentrations used for crystallization. The receptor fragment crystallizes in the orthorhombic space group P2(1)2(1)2(1), a = 105.5 A, b = 224.5 A, c = 363.5 A. The crystals are extremely radiation sensitive. Their diffraction extends to 3.8 A, and there is some diffuse scatter with helical characteristics. Analysis of these diffraction patterns indicates that the transferrin receptor fragments are arranged in continuous 8-fold symmetric helical columns parallel to the c axis, with a total of 32 receptor fragment monomers in the unit cell. A structure determination is in progress.